Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification before the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Pseudomonas aeruginosa is a Gram-negative bacterium and a major opportunistic human pathogen, accounting for approximately 15% of all hospital infections (1). Immunocompromised patients and patients with comorbid illnesses are especially susceptible to the infection (1, 2). P. aeruginosa has multiple antimicrobial resistance mechanisms making infections difficult to treat (3). High morbidity and mortality rates have been reported in P. aeruginosa infections, especially for late-onset ventilator associated pneumonia (4, 5). In P. aeruginosa, the production of virulence factors and biofilm formation are regulated by quorum sensing (QS) systems (6). QS involves bacterial cell-to-cell communication by small molecules. QS allows bacteria populations to adjust behavior in response to environmental conditions (4). Communication in QS relies on signaling molecules including the N-acyl-homoserine lactones (AHLs) found in P. aeruginosa and most other Gram-negative bacteria. AHLs are synthesized as the bacterial cell density increases. When the concentrations of AHLs reach a critical threshold, the signal molecules bind to specific receptors and regulate target genes expression. A major QS system in P. aeruginosa includes las and rhl, which use 3-oxo-C12-homoserine lactone and C4-homoserine lactone as signaling molecules, respectively. QS signaling is correlated with the virulence of P. aeruginosa infections. Deletion of single or multiple QS genes in P. aeruginosa reduced virulence in several mouse models (5). The presence of QS signaling molecules and expression of QS-responsive genes in P. aeruginosa have been detected in sputum samples of cystic fibrosis patients. And most recently, production of QS-dependent virulence factors of P. aeruginosa have been linked to the development of ventilator-associated pneumonia (6). Since inhibition of QS biosynthetic pathways does not affect cell growth, blocking QS synthesis has been proposed as a strategy to attenuate the virulence of bacterial infections without causing drug resistance (7).
AHL synthase catalyzes the production of AHL using S-adenosylmethionine (SAM) and acylated-acyl carrier protein as precursors. The reaction produces 5′-methylthioadenosine (MTA) as a product. MTA is also an important product from polyamine biosynthesis and is recycled by a SAM salvage pathway (8). In most bacteria, MTA is degraded by 5′-methylthioadenosine nucleosidase (MTAN) to adenine and 5-methylthio-α-D-ribose. Inhibition of E. coli and V. cholerae MTANs with transition state analogue inhibitors or by gene deletion, disrupts quorum sensing, and reduces biofilm formation, supporting MTAN as a target for QS in most Gram negative bacteria (8). Mammals do not express an MTAN, nor do they have QS pathways, giving species specificity to this target.
In eukaryotes and archaea, MTA degradation is catalyzed by 5′-methylthioadenosine phosphorylase (MTAP) which converts MTA and phosphate to adenine and 5-methylthio-α-D-ribose 1-phosphate (9). P. aeruginosa was originally thought to be a bacterial anomaly, possessing an MTAP (PA3004 gene) instead of MTAN. The PA3004-encoded protein was recently characterized and found to prefer methylthioinosine (MTI) as substrate (10). It remains the only known example of a specific MTI phosphorylase (MTIP). The discovery of MTIP suggested that MTA must be deaminated in P. aeruginosa. MTA catabolism in P. aeruginosa was examined using [8-14C]MTA. A MTA→MTI→hypoxanthine pathway was established and no significant MTAP or MTAN activity was observed (10). These results established a functional PaMTIP in cells and extracts and implicated the existence of an MTA deaminase (MTADA) to convert MTA to MTI (FIG. 1). If MTADA is directly and solely responsible for MTA degradation in P. aeruginosa, inhibition of PaMTADA would be functionally similar to that of MTAN in other bacterial species, causing MTA product inhibition of AHL synthase and disruption of quorum sensing in P. aeruginosa (11). This pathway is unprecedented in bacteria, but Plasmodium species also possess a similar two-step pathway of MTA degradation. In the case of Plasmodium species, both the purine nucleoside phosphorylase and the adenosine deaminase (ADA) are broad-specificity enzymes, capable of functioning as MTIP and MTADA, respectively. However, inosine and adenosine are preferred substrates and MTI and MTA are secondary substrates (12, 13).
Recently, the first specific MTA deaminase has been reported in Thermotoga martima (14). The TmMTADA can deaminate MTA, S-adenosylhomocysteine and adenosine but prefers MTA. TmMTADA was identified by using structure-based docking with high-energy forms of potential substrates and the activity validated by enzymatic assays with purified protein. A crystal structure of TmMTADA in complex with S-inosylhomocysteine, the product of SAH deamination, was determined in the same study, revealing the key residues for binding substrates in the active site (14). These findings on TmMTADA guided the search for PaMTADA.
The present invention addresses the need for compounds that attenuate the virulence of infections due to P. aeruginosa without causing drug resistance.